B and T lymphocytes recognize foreign antigen through specialized receptors: the immunoglobulins and the T cell receptor (TCR) respectively. The highly polymorphic antigen-recognition regions of these receptors are composed of variable (V), diversity (D), and joining (J) gene segments which undergo somatic rearrangement prior to their expression by a mechanism known as V(D)J recombination (Tonegawa, 1983). Each V, D, and J segment is flanked by Recombination Signal Sequences (RSSs) composed of conserved heptamers and nonamers separated by random sequences of either 12 or 23 nucleotides. RSSs serve as recognition sequences for the V(D)J Recombinase.
V(D)J recombination can be roughly divided into three steps. The RAG1 and RAG2 proteins initiate the rearrangement process through the recognition of the RSS and the introduction of a DNA double strand break (dsb) at the border of the heptamer (Schatz et al., 1989; Oettinger, 1990). RAG1 and RAG2 are the sole two factors required to catalyze DNA cleavage in cell-free systems (McBlane et al., 1995; Van Gent et al., 1995; Eastman et al., 1996) in a reaction reminiscent of retroviral integration and transposition (van Gent et al., 1996; Roth and Craig, 1998). Three acidic residues, DDE, were shown to compose the active site carried by RAG1 (Kim et al., 1999; Landree et al., 1999; Fugmann et al., 2000). The restricted expression of both RAG1 and RAG2 genes to immature B and T lymphocytes confines V(D)J recombination to the lymphoid lineage. At the end of this phase, which causes a DNA damage, the chromosomal DNA is left with two hairpin-sealed coding ends (CE), while the RSSs and the DNA intervening sequences are excised from the chromosome as blunt, phosphorylated signal ends (SE) (Roth et al., 1992; Schlissel et al., 1993; Zhu and Roth, 1995). The subsequent step consists in recognition and signaling of the DNA damage to the DNA repair machinery. From now on, ubiquitous enzymatic activities are involved.
The description of the murine scid situation, characterized by a lack of circulating mature B and T lymphocytes (Bosma et al., 1983), as a general DNA repair defect accompanied by an increased sensitivity to ionizing radiation or other agents causing DNA dsb provided the link between V(D)J recombination and DNA dsb repair (Fulop, 1990; Biedermann, 1991; Hendrickson, 1991). This was further confirmed by the analysis of Chinese ovary cell lines (CHO), initially selected on the basis of their defect in DNA repair, which turned out to have impaired V(D)J recombination in vitro (Taccioli et al., 1993). This led to the description of the Ku70/Ku80/DNA-PKcs complex as a DNA damage sensor (review in (Jackson and Jeggo, 1995)). Briefly, DNA-PKcs is a DNA-dependant protein kinase that belongs to the Phosphoinositol (PI) kinase family, which is recruited at the site of the DNA lesion through the interaction with the regulatory complex Ku70/80 that binds to DNA ends (Gottlieb and Jackson, 1993). Cells from scid mice lack DNA-PK activity owing to a mutation in the DNA-PKcs encoding gene (Blunt et al., 1996; Danska et al., 1996). This severely compromises the V(D)J recombination process, ultimately leading to an arrest in both B and T cell development.
More recently, two other proteins, NBS1 and γ-H2AX, have been identified on the site of chromosomal rearrangement in the TCR-α locus in thymocytes (Chen et al., 2000). NBS 1, which is mutated in the Nijmegen breakage syndrome, participates in the formation of the RAD50/MRE11/NBS1 complex involved in DNA repair (Carney et al., 1998; Varon et al., 1998). γ-H2AX represents the phosphorylated form of histone H2A in response to external damage and is considered as an important sensor of DNA damage (Rogakou et al., 1998; Rogakou et al., 1999; Paull et al., 2000). The biological implication of this observation is not yet fully understood, but it indicates that the RAD50/MRE11/NBS1 complex may cooperate with the DNA-PK complex in sensing and signaling the RAG1/2-mediated DNA dsb to the cellular DNA repair machinery. In the final phase of the V(D)J rearrangement, the DNA-repair machinery per se will ensure the re-ligation of the two chromosomal broken ends. This last step resembles the well-known DNA non-homologous end joining (NHEJ) pathway in the yeast Sacchaomyces cerevisiae (review in (Haber, 2000)) and involves the XRCC4 (Li et al., 1995) and the DNA-Ligase IV (Robins and Lindahl, 1996) factors. The crystal structure recently obtained for XRCC4 demonstrates the dumb-bell like conformation of this protein and provides a structural basis for its binding to DNA as well as its association with DNA-Ligase IV (Junop et al., 2000). All the animal models carrying a defective gene of either one of the known V(D)J recombination factors, either natural (murine and equine scid) or engineered through homologous recombination, have a profound defect in the lymphoid developmental program owing to an arrest of the B and T cell maturation at early stages (Mombaerts et al., 1992; Shinkai et al., 1992; Nussenzweig et al., 1996; Zhu et al., 1996; Jhappan et al., 1997; Shin et al., 1997; Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998; Gao et al., 1998; Taccioli et al., 1998). In the cases of DNA-LigaseIV and XRCC4 this phenotype is also accompanied by an early embryonic lethality caused by massive apoptotic death of postmitotic neurons (Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998).
In humans, several immune deficiency conditions are characterized by faulty T and/or B cell developmental program (Fischer et al., 1997). In about 20% of the cases, the severe combined immunodeficiency (SCID) phenotype is caused by a complete absence of both circulating B and T lymphocytes, associated with a defect in the V(D)J recombination process, while Natural Killer (NK) cells are present. Mutations in either the RAG1 or RAG2 gene account for a subset of patients with this condition (Schwarz et al., 1996; Comeo et al., 2000; Villa et al., 2001). In some patients (RS-SCID), the T-B-SCID defect is not caused by RAG1 or RAG2 mutations and is accompanied by an increased sensitivity to ionizing radiations of both bone marrow cells (CFU-GMs) and primary skin fibroblasts (Cavazzana-Calvo et al., 1993), as well as a defect in V(D)J recombination in fibroblasts (Nicolas et al., 1998).
Although this condition suggests that RS-SCID could have a general DNA-repair defect reminiscent of the murine scid situation, DNA-PK activity was found normal in these patients and the implication of the DNA-PKcs gene has been unequivocally ruled out by genetic means in several consanguineous families (Nicolas et al., 1996). A role for all the other known genes involved in V(D)J recombination/DNA repair was equally excluded as being responsible for RS-SCID condition (Nicolas et al., 1996). The gene defective in RS-SCID therefore encodes a yet undescribed factor. The inventors recently assigned the disease related locus to the short arm of human chromosome 10, in a 6.5 cM region delimited by two polymorphic markers D10S1664 and D10S674 (Moshous et al., 2000), a region shown to be linked to a similar SCID condition described in Athabascan speaking American Indians (A-SCID) (Hu et al., 1988; Li et al., 1998).